This invention generally relates to methods for delivery of an active agent to a subject and more specifically to the use of electroporation and needle-free delivery of an active agent to a subject.
A cell has a natural resistance to the passage of molecules through its membranes into the cell cytoplasm. Scientists in the 1970""s first discovered xe2x80x9celectroporation,xe2x80x9d the use of electrical fields to create pores in cells without causing permanent damage to the cells. This discovery made possible the insertion of large molecules directly into cell cytoplasm. Electroporation was further developed to aid in the insertion of various molecules into cell cytoplasm by temporarily creating pores in the cells through which the molecules pass into the cell.
Electroporation has been used in both in vitro and in vivo procedures to introduce foreign material into living cells. With in vitro applications, a sample of live cells is first mixed with the agent to be introduced therein and placed between electrodes, such as parallel plates. Then, the electrodes are used to apply an electrical field to the mixture containing the cells and the agent to be introduced therein.
With in vivo applications of electroporation, electrodes are provided in various configurations such as, for example, a caliper that grips the epidermis overlying a region of cells to be treated. Alternatively, needle-shaped electrodes may be inserted into the patient, to access more deeply located cells. In either case, before, simultaneously, or after the agent is injected into the treatment region, the electrodes are used to apply an electrical field to the region. See, for example, U.S. Pat. No. 5,019,034, issued May 28, 1991 and U.S. Pat. No. 5,702,359, issued Dec. 30, 1997.
Electroporation (both in vitro and in vivo) functions by causing cell membranes to which a brief high voltage pulse is administered to temporarily become porous, whereupon molecules can enter the cells. In some electroporation applications, the electric field comprises a single square wave pulse on the order of 1000 V/cm, of about 100 xcexcs duration. Such a pulse may be generated, for example, in known applications of the ElectroSquarePorator T820, made by the BTX Division of Genetronics, Inc.
Electroporation has been recently suggested as an alternate approach to the treatment of certain diseases such as cancer by introducing a chemotherapeutic drug directly into the cell. For example, in the treatment of certain types of cancer with chemotherapy it is necessary to use a high enough dose of a drug to kill the cancer cells without killing an unacceptably high number of normal cells. If the chemotherapy drug could be inserted directly inside the cancer cells, this objective could be achieved. However, some of the best anti-cancer drugs, for example, bleomycin, cannot penetrate the membranes of certain cancer cells effectively under normal circumstances. To overcome this difficulty, electroporation has been used to cause bleomycin to penetrate the membranes of cancer cells.
Electroporation-assisted chemotherapy typically is carried out by injecting an anticancer drug directly into the tumor and applying an electric field to the tissue between a pair of electrodes. The field strength must be adjusted reasonably accurately so that electroporation of tumor cells occurs without damage, or at least with minimal damage, to any normal or healthy cells. Typically, this method is employed with tumors located on the exterior of the patient""s body by applying electrodes to the body surface on opposite sides of the tumor, thus creating an electric field between the electrodes. When the field is uniform, the distance between the electrodes can then be measured and a suitable voltage, derived according to the formula E=V/d (wherein E=electric field strength in V/cm; V=voltage in Volts; and d=distance in cm), can then be applied to the electrodes . However, when the tumors to be treated are large, irregular in shape, or located within the body interior, it is more difficult to properly locate electrodes and measure the distance between them so as to accurately calculate the voltage that is to be applied. In such cases, needle array electrodes as, for example, described in U.S. Pat. No. 5,993,434 (Dev and Hofmann) have proven to be advantageous.
Using these and related techniques (for example, the molecule can be delivered encapsulated in a liposome), electroporation has been used to deliver molecules into many different types of cells. For example, electroporation has been used to deliver biologically active agents to various human and mammalian cells, such as egg cells (i.e., oocytes), sperm, platelets, muscle, liver, skin, and red blood cells. In addition, electroporation has been used to deliver molecules to plant protoplasts, plant pollen, bacteria, fungi, and yeast. A variety of different biologically active molecules and agents have been delivered to cells using this technique, including DNA, RNA and various chemical agents.
The first hypodermic syringe was developed by a French surgeon, Charles Gabriel Pravaz, in 1853 to take advantage of the highly permeable interstitial tissue below the skin surface to transport pharmaceuticals to active sites. Although there have been developments in hypodermic syringes since then, the technology has remained essentially unchanged for the past 150 years. Needle-free injection was developed when workers on hydraulic equipment noticed that high-pressure squirts of hydraulic oil would pierce the skin. The first description of needle-free injection was in Marshall Lockhart""s 1936 patent for xe2x80x9cjet injection.xe2x80x9d Then, in the early 1940""s Higson and others developed high-pressure xe2x80x9cgunsxe2x80x9d using a very fine jet of liquid medicament to pierce the skin and deposit it into the tissue underneath. In World War II, needle-free guns were used extensively to inoculate troops en masse against infectious disease. Later, needle-free guns were applied more generally in large-scale vaccination programs.
However, these early needle-free injectors were used on multiple patients and fears about the transmission of hepatitis B and HIV infection by reuse of the injectors led to a sharp decline in their use. Until recently, the main application of such devices was veterinary, with a few being used by diabetics for self-treatment.
In the past 50 years, over 300 patents have been filed in the needle-free delivery area. Although various improved products have come to the market, none has gained wide use and remnants of the older devices remain to this day. These devices tend to be expensive to purchase and difficult to use, requiring the user to perform a series of complicated steps to set up the device for use. For example, some of these systems require the user to fit a needle to the delivery device temporarily in order to draw liquid containing the desired active agent into the device from a vial. Therefore, even the more modem needle-free delivery systems do not address the needs of the market for an easy to use, low cost, and simple system. Consequently, needle-free delivery has not come into widespread use.
Despite this apparent failure of needle-free delivery, the pharmacokinetics and pharmacodynamics of needle-free delivery are well documented. Accelerating a jet of liquid to high speed provides power for the liquid to penetrate the stratum corneum as well as individual cell membranes. Thus, there is a need in the art for new and better methods for transporting molecules, such as biologically active agents, across the stratum corneum and/or cell membranes in treatment of a variety of conditions and diseases.
The present invention overcomes such problems in the art by providing methods for introducing biologically active agents into cells without use of a hypodermic needle. In one embodiment according to the present invention, a biologically active agent is introduced in a form suitable for direct or indirect electrotransport into a region of tissue of the subject using one or more needle-free injectors, and an electric field is applied to the region of tissue, thereby causing electroporation of the region of tissue prior to, simultaneously with, and/or subsequently to introducing the agent. Direct electrotransport refers to the transport of molecules subjected to an electrical or magnetic force, indirect electrotransport refers to the transport of molecules facilitated by electric forces which act primarily on transport barriers, e.g., cell membranes, which become more permeable as a result of electric forces. The combination of needle-free injection and electroporation is sufficient to introduce the active agent into the cell and allows for delivery of pharmaceutical compounds, nucleic acid constructs, or other agents into cells contained within the tissue region so treated.
In another embodiment according to the present invention, a biologically active agent is introduced into cells in a region of tissue of a subject by contacting the region of tissue or adjacent tissue with two or more spaced apart needle-free injectors while injecting a biologically active agent into the tissue, and applying an electrical field to the tissue via the two or more injectors prior to, simultaneously with, and/or subsequently to injection of the agent so as to electroporate the region of tissue, whereby the combination of needle-free injection and electroporation is sufficient to introduce the agent into the cell.
In yet another embodiment according to the present invention, a biologically active agent is introduced into cells in a region of tissue of a subject by contacting the region of tissue with at least one needle-free injector while injecting an agent suitable for direct or indirect electrotransport into the region of tissue, and applying an electrical field across the region of tissue using the at least one injector prior to, simultaneously with, and/or subsequently to injection of the agent, whereby the combination of needle-free injection and electroporation is sufficient to introduce the agent into the cell.